KU7 Cells Research Articles

Deglycosylation Does Not Affect the Cytotoxic Action of Adenoviral Interferon α‐Induced Bystander Proteins

We previously reported an indirect effect of adenoviral interferon α (Ad‐IFNα) that is mediated by secreting bystander proteins (BSPs) and causing cytotoxic responses in both interferon α (IFNα)‐sensitive and –resistant cancer cells. Here, we investigated whether glycosylation of BSPs is necessary for the inhibition of IFNα‐resistant human bladder cancer cell line (KU7). The condition medium (CM) from the normal human urothelial cell line, TERT‐NHUC, was collected as described previously and incubated with and without N‐glycosidase F at 37°C (overnight). The growth inhibition of KU7 was compared between the control and experimental conditions. The proliferation of KU7 cells treated with interferon a protein (Intron A) is comparable to the cells treated with control TERT‐NHUC‐CM (without Ad‐IFNα infection) after 72 hr incubation. The growth of KU7 was significantly inhibited by the CM from Ad‐IFNα–infected TERT‐NHUC culture. N‐glycosidase F incubation effectively deglycosylated the BSPs in the CM (Ad‐IFNα TERT‐NHUC) as demonstrated by the band shifting in the PAGE gel verification. The deglycosylated CM (Ad‐IFNα TERT‐NHUC) produced a same degree of inhibition on the proliferation of KU7 cells (Fig 1). In conclusion, the cytotoxic effects of BSPs is independent of its status of glycosylation.

MicroRNA-145 promotes differentiation in human urothelial carcinoma through down-regulation of syndecan-1.

BackgroundA new molecular marker of carcinoma in the urinary bladder is needed as a diagnostic tool or as a therapeutic target. Potential markers include microRNAs (miRNAs), which are short, low molecular weight RNAs 19–24 nt long that regulate genes associated with cell proliferation, differentiation, and development in various cancers. In this study, we investigated the molecular mechanisms by which miR-145 promotes survival of urothelial carcinoma cells and differentiation into multiple lineages. We found miR-145 to regulate expression of syndecan-1, a heparin sulfate proteoglycan.MethodsCell proliferation in the human urothelial carcinoma cell lines T24 and KU7 was assessed by MTS assay. Cellular senescence and apoptosis were measured by senescence-associated β-galactosidase (SA-β-gal) and TUNEL assay, respectively. Quantitative RT-PCR was used to measure mRNA expression of various genes, including syndecan-1, stem cell factors, and markers of differentiation into squamous, glandular, or neuroendocrine cells.ResultsOverexpression of miR-145 induced cell senescence, and thus significantly inhibited cell proliferation in T24 and KU7 cells. Syndecan-1 expression diminished, whereas stem cell markers such as SOX2, NANOG, OCT4, and E2F3 increased. miR-145 also up-regulated markers of differentiation into squamous (p63, TP63, and CK5), glandular (MUC-1, MUC-2, and MUC-5 AC), and neuroendocrine cells (NSE and UCHL-1). Finally, expression of miR-145 was down-regulated in high-grade urothelial carcinomas, but not in low-grade tumors.ConclusionsResults indicate that miR-145 suppresses syndecan-1 and, by this mechanism, up-regulates stem cell factors and induces cell senescence and differentiation. We propose that miR-145 may confer stem cell-like properties on urothelial carcinoma cells and thus facilitate differentiation into multiple cell types.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1846-0) contains supplementary material, which is available to authorized users.

Abstract 1922: Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression.

Abstract Introduction: Bladder cancer (BCa) is among the five most common malignancies worldwide and has the highest recurrence rate of any tumor type. BCa biology is poorly understood and early detection remains one of the most urgent issues in BCa research. Previously we have identified a preliminary molecular signature of BCa in voided urine samples from BCa patients. Our results revealed aberrant overexpression of the serine protease inhibitor (serpin) plasminogen activator inhibitor type-1 (PAI-1), both at the genomic and protein level. Increasing evidence indicate that elevated levels of PAI-1 correlates to a poor prognosis in many tumor types including breast, colorectal, gastric, ovarian, and more recently bladder cancer. PAI-1 has emerged as a potential target for BCa treatment and the aim of this study is to provide a greater understanding of its role in bladder cancer progression. Methods: PAI-1 expression levels in UROtsa (benign urothelial cell), KU7 (low-grade papillary urothelial cancer cell), and UMUC14 and T24 (high-grade invasive urothelial cancer cell lines) was tested by real-time PCR (RTPCR) and Western blot analysis. Secreted PAI-1 levels were measured by ELISA. KU7 stable clones overexpressing PAI-1 was established by transfection of plasmid DNA encoding human PAI-1. T24 and UMUC14 stable clones with shRNA knockdown of PAI-1 were established by infection with retroviral particles. Cell proliferation, migration, invasion, and apoptosis assays were performed in stable clones. Results: RTPCR analysis revealed an increase of PAI-1 in T24 and UMUC14 cells (27.9 and 5.2 times, respectively) while KU7 cells exhibited significantly reduced levels as compared to the UROtsa control. Similar results were observed for PAI-1 protein concentrations in cell culture supernatants and western blot analysis. While overexpression of PAI-1 in KU7 cells promoted an increase in proliferation, migration and invasion, these were significantly reduced upon inactivation of PAI-1 in T24 and UMUC14 cells stably expressing shRNA PAI-1. Loss of PAI-1 function also led to a substantial increase in apoptotic cell death. Conclusions: These results suggest that PAI-1 overexpression in BCa promotes tumor progression and therapeutic strategies targeting PAI-1 may serve clinically beneficial outcomes. Funding provided by research grants from Florida Department of Health James and Esther King Team Science Award 10KT-01 (CJR) and National Cancer Institute RO1 CA116161 (SG). Citation Format: Evan Gomes, Steven Goodison, Makito Miyake, Shanti Ross, Ge Zhang, Charles Rosser. Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2013-1922

ALKBH2, a novel AlkB homologue, contributes to human bladder cancer progression by regulating MUC1 expression

The ALKBH family of proteins are highly expressed in various types of human cancer where they are involved in tumor growth and progression. However, multiple isoforms of ALKBH exist and the effect of individual isoforms on the development of urinary bladder cancer is unknown, particularly the molecular mechanisms involved in the progression from a noninvasive to invasive phenotype. We examined the role and function of ALKBH2 in human bladder cancer development in vitro and provide the first report that suppression of ALKBH2 in a human urothelial carcinoma cell line, KU7, reduces the expression of the transmembrane mucin protein, MUC1, and induces G1 cell cycle arrest. Moreover, reduction of ALKBH2 suppressed epithelial to mesenchymal transition (EMT) via increasing E-cadherin and decreasing vimentin expression. Transfection of MUC1 siRNA inhibited cell proliferation and EMT to the same extent as ALKBH2 gene silencing in vitro. ALKBH2 knockdown significantly suppressed MUC1 expression and tumor volume of bladder cancers in vivo as assessed in an orthotopic mouse model using ALKBH2 shRNA transfected KU7 cells. Immunohistochemical examination showed high expression levels of ALKBH2 in human urothelial carcinoma samples, especially in high-grade, superficially and deeply invasive carcinomas (pT(1) and >pT(2)), and in carcinoma in situ but not in normal urothelium. This study demonstrates that ALKBH2 is an upstream molecule of the oncoprotein, MUC1, and regulates cell cycle and EMT, resulting in progression of urothelial carcinomas.

Abstract 1452: mTOR inhibition radiosensitizes bladder cancer tumor cells in vitro and in vivo: A novel strategy for treatment

Abstract Background: Radiation therapy for invasive bladder cancer allows for organ preservation but systemic toxicity and local control remain problematic. As such, there is a need to increase radiosensitization of tumor cells to improve efficacy. The aim of this study was to investigate if mTOR (mammalian target of rapamycin), a downstream kinase of the PI3K/Akt survival pathway, may be a target for combined bladder cancer therapy. Methods: Clonogenic assays were performed using 6 bladder cancer cell lines in order to address the effects of ionizing radiation (IR) on growth, when tested alone and in combination with RAD001, a potent mTOR inhibitor. Cell cycle analysis was performed using flow cytometry. In the in vivo study, nude mice were subcutaneously injected with KU7 and 253J-BV cells. Treatment with RAD001 (1.5 mg/kg, daily), fractionated IR (total 9 Gy), and the combination of RAD001 and IR was followed over 4 weeks. Tumor growth kinetics was measured and tumor weight at the experimental endpoint. Results: In vitro, a significant decrease in colony formation was observed in the combined treatment when compared to RAD001 or radiation alone (p<0.05) in all cell lines. A G0/G1 as well as a significant increase in G2 arrest was observed in the combined treatment compared to either treatment alone. Changes in the levels of Cyclin D1, p27 and p21 correlated with the observed changes in the cell cycle. Moreover, IR rapidly activated AKT whereas RAD001 effectively inhibited the mTOR downstream signaling as shown by the inhibition of the phosphorylation of S6. Furthermore, autophagy was induced following the treatment with RAD001 and in combination as indicated by the conversion of LC3-I to LC3-II, a protein marker for autophagy. Our in vivo data confirmed our in vitro data, wherein a significant decrease in tumor weight was observed in the combined treatment arm (90% decrease, p<0.001) compared to either treatment alone (60% decrease for RAD001, p<0.05; 77% decrease for IR, p<0.05). These findings point to additive and possibly synergistic beneficial effects of the combined on bladder cancer. Conclusions: The inhibition of mTOR signaling appears promising as a therapeutic modality for bladder cancer, especially in the context of combination with radiation therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1452. doi:1538-7445.AM2012-1452

Celastrol decreases specificity proteins (Sp) and fibroblast growth factor receptor-3 (FGFR3) in bladder cancer cells

Celastrol (CSL) is a naturally occurring triterpenoid acid that exhibits anticancer activity, and in KU7 and 253JB-V bladder cells, CSL induced apoptosis, inhibited growth, colony formation and migration and CSL decreased bladder tumor growth in vivo. CSL also decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several Sp-regulated genes/proteins including vascular endothelial growth factor, survivin and cyclin D1 and fibroblast growth factor receptor-3, a potential drug target for bladder cancer therapy, has now been characterized as an Sp-regulated gene downregulated by CSL. The mechanism of Sp downregulation by CSL was cell context-dependent due to activation of proteosome-dependent (KU7) and -independent (253JB-V) pathways. In 253JB-V cells, CSL induced reactive oxygen species (ROS) and inhibitors of ROS blocked CSL-induced growth inhibition and repression of Sp1, Sp3 and Sp4. This response was due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of miR-27a and miR-20a/17-5p, respectively, which regulate expression of these transcriptional repressors. Thus, the anticancer activity of CSL in 253JB-V cells is due to induction of ROS and ROS-mediated induction of Sp repressors (ZBTB4/ZBTB10) through downregulation of miR-27a and miR-20a/17-5p.

In vivo Evaluation of Mucoadhesive Nanoparticulate Docetaxel for Intravesical Treatment of Non–Muscle-Invasive Bladder Cancer

The present work describes the development and in vitro and in vivo evaluation of a mucoadhesive nanoparticulate docetaxel (DTX) formulation for intravesical bladder cancer therapy. Mucoadhesive formulations based on hyperbranched polyglycerols (HPG), hydrophobically derivatized with C(8)/C(10) alkyl chains in the core and modified with methoxy-polyethylene glycol (MePEG) and amine groups in the shell (HPG-C(8/10)-MePEG-NH(2)) were synthesized and DTX was loaded into these by a solvent evaporation method. Both low-grade (RT4, MGHU3) and high-grade (UMUC3) human urothelial carcinoma cell lines were treated with various concentrations of DTX formulations in vitro. KU7 cells that stably express firefly luciferase (KU7-luc) were inoculated in female nude mice by intravesical instillation and quantified using bioluminescence imaging. Mice with established KU7-luc tumors were given a single intravesical instillation with PBS, Taxotere (DTX from Sanofi-aventis), and DTX-loaded HPG-C(8/10)-MePEG and/or HPG-C(8/10)-MePEG-NH(2). Drug uptake was conducted using LC/MS-MS (liquid chromatography/tandem mass spectrometry) and tumor microenvironment and uptake of rhodamine labeled HPGs was assessed. In vitro, all DTX formulations potently inhibited bladder cancer proliferation. However, in vivo, DTX-loaded HPG-C(8/10)-MePEG-NH(2) (mucoadhesive DTX) was the most effective formulation to inhibit tumor growth in an orthotopic model of bladder cancer. Furthermore, mucoadhesive DTX significantly increased drug uptake in mouse bladder tissues. In addition, rhodamine labeled HPG-C(8/10)-MePEG-NH(2) showed enhanced uptake of these nanoparticles in bladder tumor tissues. Our data show promising in vivo antitumor efficacy and provide preclinical proof of principle for the intravesical application of mucoadhesive nanoparticulate DTX formulation in the treatment of bladder cancer.

The use of nanocrystalline cellulose for the binding and controlled release of drugs

The objective of this work was to investigate the use of nanocrystalline cellulose (NCC) as a drug delivery excipient. NCC crystallites, prepared by an acid hydrolysis method, were shown to have nanoscopic dimensions and exhibit a high degree of crystallinity. These crystallites bound significant quantities of the water soluble, ionizable drugs tetratcycline and doxorubicin, which were released rapidly over a 1-day period. Cetyl trimethylammonium bromide (CTAB) was bound to the surface of NCC and increased the zeta potential in a concentration-dependent manner from −55 to 0 mV. NCC crystallites with CTAB-modified surfaces bound significant quantities of the hydrophobic anticancer drugs docetaxel, paclitaxel, and etoposide. These drugs were released in a controlled manner over a 2-day period. The NCC-CTAB complexes were found to bind to KU-7 cells, and evidence of cellular uptake was observed.

Direct gene transfer of adenoviral-mediated interferon α into human bladder cancer cells but not the bystander factors produced induces endoplasmic reticulum stress-related cytotoxicity

We have previously shown that adenoviral-mediated interferon α (Ad-IFNα) is cytotoxic both to cells that are sensitive to recombinant interferon α (IFNα) and to cells which are resistant to IFNα. The cancer cell-specific cytotoxic effects of Ad-IFNα involve three different mechanisms: 1. The direct effect of IFNα production causing cancer cell death in IFNα sensitive cells (1); 2. The direct effect of Ad-IFNα infection and high levels of IFNα expression in IFNα resistant cancer cells (2); and 3. The indirect effect of the Ad-IFNα bystander factors produced (2-4). After Ad-IFNα infection, the cells produce a large amount of perinuclear localized IFN protein. This protein over-load could be a major factor in the direct cancer cell death of those cells infected with Ad-IFNα compared with the indirect cytotoxic effects of the bystander factors produced. Here, we investigated whether a component of Ad-IFN-induced cell death involves protein overload-induced endoplasmic reticulum (ER) stress, using an IFNα-resistant human bladder cancer cell line (KU7), and the normal human urothelial cell line, TERT-NHUC, as preclinical models. We found that the two ER stress response pathways examined were activated in KU7 cells. In contrast, following treatment of the normal TERT-NHUC cells with Ad-IFNα, no ER stress signals were observed. In addition, no ER stress related changes were seen, when KU7 cells were exposed to conditioned medium from Ad-IFNα-treated KU7 cells, indicating that bystander produced cytotoxicity did not involve ER stress. After 24 h of Ad-IFNα infection, the KU7 cancer cells produced spliced X-box binding protein 1 and activating transcription factor 6 protein (ATF6), evoking an ER stress response that could contribute to Ad-IFNα induced apoptosis in these cancer cells. In addition, GADD153/CHOP, GADD34 and BAX were also subsequently modified following activation of the ER stress pathways, thereby signaling downstream effectors in a pro-apoptotic manner.

Development and in vitro characterization of paclitaxel and docetaxel loaded into hydrophobically derivatized hyperbranched polyglycerols

In this study we report the development and in vitro characterization of paclitaxel (PTX) and docetaxel (DTX) loaded into hydrophobically derivatized hyperbranched polyglycerols (HPGs).Several HPGs derivatized with hydrophobic groups (C8/10 alkyl chains) (HPG–C8/10–OH) and/or methoxy polyethylene glycol (MePEG) chains (HPG–C8/10–MePEG) were synthesized. PTX or DTX were loaded into these polymers by a solvent evaporation method and the resulting nanoparticle formulations were characterized in terms of size, drug loading, stability, release profiles, cytotoxicity, and cellular uptake.PTX and DTX were found to be chemically unstable in unpurified HPGs and large fractions (∼80%) of the drugs were degraded during the preparation of the formulations. However, both PTX and DTX were found to be chemically stable in purified HPGs. HPGs possessed hydrodynamic radii of less than 10nm and incorporation of PTX or DTX did not affect their size. The release profiles for both PTX and DTX from HPG–C8/10–MePEG nanoparticles were characterized by a continuous controlled release with little or no burst phase of release. In vitro cytotoxicity evaluations of PTX and DTX formulations demonstrated a concentration-dependent inhibition of proliferation in KU7 cell line. Cellular uptake studies of rhodamine-labeled HPG (HPG–C8/10–MePEG13–TMRCA) showed that these nanoparticles were rapidly taken up into cells, and reside in the cytoplasm without entering the nuclear compartment and were highly biocompatible with the KU7 cells.

Arsenic trioxide downregulates specificity protein (Sp) transcription factors and inhibits bladder cancer cell and tumor growth

Arsenic trioxide exhibits antiproliferative, antiangiogenic and proapoptotic activity in cancer cells, and many genes associated with these responses are regulated by specificity protein (Sp) transcription factors. Treatment of cancer cells derived from urologic (bladder and prostate) and gastrointestinal (pancreas and colon) tumors with arsenic trioxide demonstrated that these cells exhibited differential responsiveness to the antiproliferative effects of this agent and this paralleled their differential repression of Sp1, Sp3 and Sp4 proteins in the same cell lines. Using arsenic trioxide-responsive KU7 and non-responsive 253JB-V bladder cancer cells as models, we show that in KU7 cells, ≤ 5 μM arsenic trioxide decreased Sp1, Sp3 and Sp4 and several Sp-dependent genes and responses including cyclin D1, epidermal growth factor receptor, bcl-2, survivin and vascular endothelial growth factor, whereas at concentrations up to 15 μM, minimal effects were observed in 253JB-V cells. Arsenic trioxide also inhibited tumor growth in athymic mice bearing KU7 cells as xenografts, and expression of Sp1, Sp3 and Sp4 was significantly decreased. Inhibitors of oxidative stress such as glutathione or dithiothreitol protected KU7 cells from arsenic trioxide-induced antiproliferative activity and Sp repression, whereas glutathione depletion sensitized 253JB-V cells to arsenic trioxide. Mechanistic studies suggested that arsenic trioxide-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide.

Abstract 1502: Adenoviral-mediated interferon α induces endoplasmic reticulum stress related cytotoxicity in human cancer but not normal cells

Abstract We have previously shown that adenoviral-mediated interferon α (Ad-IFNα) treatment is specifically cytotoxic to various cancer cells, not only to the cancer cells sensitive to the interferon protein (IFNα), but also to the cells which are resistant to IFNα. We also found that there is very little cytotoxic effect of Ad-IFNα on non-cancer cells. The cancer cell specific toxic effects of Ad-IFNα can be classified into three different mechanisms: 1. Expression and secretion of interferon α protein that results in kill of IFNα sensitive cancer cells; 2. Direct kill of IFNα resistant cancer cells by Ad-IFNα and 3. Cancer cell kill by Ad-IFNα produced bystander factors. After Ad-IFNα infection, the host cells produce a large amount of IFN protein. We hypothesized that this protein over-load is cytotoxic to cancer cells and may be a major reason for Ad-IFNα direct effect. We now report that Ad-IFNα infection induces ER stress and ER stress related apoptosis in human cancer but not normal urothelial cells. In addition we found that the Ad-IFNα produced bystander cytotoxicity was not related to ER stress. To investigate whether Ad-IFNα induced cancer specific cytotoxicity was correlated with the activation of ER stress pathways, the expression of several markers of ER stress was studied. Experiments utilized the interferon resistant human bladder cancer cell line, KU7, and the normal human urothelial cell line, TERT-NHU, for the studies. We found that three ER stress response pathways examined were activated in KU7 cells. In contrast, the ER stress response pathways remained silent in the normal TERT-NHU cells or in KU7 cells treated with conditioned medium from Ad-IFNα treated KU7 cells (to test the effect of bystander factor produced cytotoxicity) the ER stress response pathways remained silent. After 24hr of Ad-IFNα exposure, KU7 cancer cells produced spliced X-box binding protein 1 (sXBP1), activating transcription factor 4 (ATF4) and activating transcription factor (ATF6) protein, and evoked an ER stress response that contribute to Ad-IFNα induced apoptosis in cancer cells. In addition, we found GADD153/CHOP and GADD34 were also upregulated following the activation of the three ER stress pathways, thereby signaling downstream effectors in a pro-apoptotic manner. Ad-IFNα recently has been used in Phase I clinical trial for patient with superficial bladder cancer. The results presented here could provide a mechanistic basis for Ad-IFNα action and support the positive clinical results obtained to date. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1502.

784 SYNERGISTIC THERAPEUTIC EFFECT OF NF-κB AND AP-1 FUNCTION INHIBITORS ON SUPPRESSION OF CELL GROWTH AGAINST HIGHLY AGGRESSIVE BLADDER CANCER CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2010784 SYNERGISTIC THERAPEUTIC EFFECT OF NF-κB AND AP-1 FUNCTION INHIBITORS ON SUPPRESSION OF CELL GROWTH AGAINST HIGHLY AGGRESSIVE BLADDER CANCER CELLS Eriko Suzuki, Eiji Kikuchi, Yutaka Horiguchi, and Mototsugu Oya Eriko SuzukiEriko Suzuki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Yutaka HoriguchiYutaka Horiguchi More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1433AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES NF-κB is often constitutively activated in bladder carcinoma cells, which may cause difficulty in treatment. AP-1 may also increase malignant character. In the present study we assessed the cytotoxic effect of a novel NF-κB inhibitor (dehydroxymethyl epoxyquinomycin, DHMEQ) and/or a strong AP-1 inhibitor (DTCM-glutarimide, DTCM-G) against bladder cancer cells with various levels of NF-κB and AP-1 activity. METHODS Activation of either NF-κB or AP-1 was examined by EMSA in 7 bladder cancer cell lines (KU-7, KU-19-19, T24, 5637, UMUC-3, RT-4 and MBT-2). NF-κB and AP-1 activities were evaluated by phosphorylation of p65 and c-Jun, respectively by Western blotting analysis. Cell proliferation was assessed by MTT assay. RESULTS Among 7 bladder cancer cell lines tested, KU-7 and KU-1919 cells revealed constitutive activation of NF-κB, while 3 including KU-1, KU-7 and T24 cells revealed constitutive activation of AP-1. Especially KU-7 cells showed potent NF-κB and AP-1 activities. The binding of NF-κB to DNA was completely inhibited after exposure to 10 μg/mL of DHMEQ for 2 to 6 hr in KU-7 and KU-19-19 cells. These cell viabilities were significantly inhibited by DHMEQ in a dose-dependent manner. DTCM-G inhibited the DNA binding activity of AP-1 for 3 to 12 hr in KU-7 and T24 cells. The combination treatment of DHMEQ (10 μg/ml) with DTCM-G (10 μg/ml) resulted in a significantly higher (56%) inhibition of cell proliferation compared to treatment with DHMEQ (30%) alone or DTCM-G (32.4%) alone (p<0.001 for both). The synergistic effects were not observed in either KU-19-19 or T24 cells. Significant level of apoptosis was induced by both DHMEQ and DTCM-g when cells were treated in combination with Docetaxel. Compared to normal tissues, clinical samples of cancer tissues from patients with invasive bladder cancer revealed higher levels of AP-1 and NF-κB activity. CONCLUSIONS Aggressive bladder carcinoma tissues were found to show high NF-κB and AP-1 activities. Combination of DHMEQ and newly discovered AP-1 inhibitor DTCM-G synergistically inhibited the growth of KU-7 bladder carcinoma cells. Dual blockade of cellular NF-κB and AP-1 activities would be a novel approach for the treatment against highly aggressive bladder cancer. Tokyo, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e307 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eriko Suzuki More articles by this author Eiji Kikuchi More articles by this author Yutaka Horiguchi More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Activation of Nerve Growth Factor-Induced Bα by Methylene-Substituted Diindolylmethanes in Bladder Cancer Cells Induces Apoptosis and Inhibits Tumor Growth

Nerve growth factor-induced B (NGFI-B) genes are orphan nuclear receptors, and NGFI-B alpha (Nur77, TR3) is overexpressed in bladder tumors and bladder cancer cells compared with nontumorous bladder tissue. 1,1-Bis(3'-indolyl)-1-(p-methoxyphenyl)-methane (DIM-C-pPhOCH(3)) and 1,1-bis(3'-indolyl)-1-(p-phenyl)methane have previously been identified as activators of Nur77, and both compounds inhibited growth and induced apoptosis of UC-5 and KU7 bladder cancer cells. The proapoptotic effects of methylene-substituted diindolylmethanes (C-DIMs) were unaffected by cotreatment with leptomycin B and were dependent on nuclear Nur77, and RNA interference with a small inhibitory RNA for Nur77 (iNur77) demonstrated that C-DIM-induced activation of apoptosis was Nur77-dependent. Microarray analysis of DIM-C-pPhOCH(3)-induced genes in UC-5 bladder cancer cells showed that this compound induced multiple Nur77-dependent proapoptotic or growth inhibitory genes including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), cystathionase, p21, p8, and sestrin-2. DIM-C-pPhOCH(3) (25 mg/kg/d) also induced apoptosis and inhibited tumor growth in athymic nude mice bearing KU7 cells as xenografts, demonstrating that Nur77-active C-DIMs exhibit potential for bladder cancer chemotherapy by targeting Nur77, which is overexpressed in this tumor type.

Measuring soluble forms of extracellular cytokeratin 18 identifies both apoptotic and necrotic mechanisms of cell death produced by adenoviral-mediated interferon α: possible use as a surrogate marker

Adenoviral transduction of human bladder cancer cells with human interferon α-2b (Ad-IFN) produces cancer specific cell death via direct and indirect mechanisms. The indirect mechanisms involve the secreted IFN produced which kill IFN protein sensitive cancer cells as well as yet unidentified bystander factors which are cytotoxic to neighboring cancer cells. The direct cell kill results from transfection and expression of Ad-IFN in the cancer cells. Since the molecular forms of cytokeratin 18 (CK18), either caspase cleaved or not, have been associated with apoptotic or necrotic cell death, respectively, we determined if increases in either or both CK18 forms could be observed following IFNα protein or Ad-IFN treatment of bladder carcinoma cells. Quantification of M30 and M65 ELISAs (assays for CK18 associated apoptotic and necrotic cell death, respectively) were used as surrogate markers of the cell death produced. In the IFN protein sensitive RT4 bladder cancer cells IFN produced primarily M30 related cell death whereas Ad-IFN treatment resulted in high levels of both M30 and M65. In contrast, conditioned medium from Ad-IFN treated cells whether from normal human urothelial cells or bladder cancer cells caused mainly increases in M30 levels when added to IFN protein resistant KU7 or UC9 bladder cancer cells, suggesting that the bystander factors present in the CM produced primarily apoptotic cell death. In addition, a significant increase in M65 levels above that observed for M30 was seen when the IFN protein resistant KU7 and UC9 cells were treated with Ad-IFN, again indicating there is additional necrotic related cell death produced by Ad-IFN as well. Normal urothelial cells showed no cytotoxicity nor increases in M30 or M65 following Ad-IFN treatment. Since intravesical Ad-IFN treatment is presently being evaluated for its efficacy in superficial bladder cancer that measurement of M30 and M65 levels in the urine at various time-points before and after Ad-IFN treatment may provide not only a biomarker of efficacy but also evidence for the different types and proportion of cell kill produced by the various mechanisms of cell kill in the tumors of individual patients.

Curcumin decreases specificity protein expression in bladder cancer cells.

Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. In this study, 10 to 25 micromol/L curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Because expression of survivin, VEGF, and VEGFR1 are dependent on specificity protein (Sp) transcription factors, we also investigated the effects of curcumin on Sp protein expression as an underlying mechanism for the apoptotic and antiangiogenic activity of this compound. The results show that curcumin induced proteasome-dependent down-regulation of Sp1, Sp3, and Sp4 in 253JB-V and KU7 cells. Moreover, using RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4, we observed that curcumin-dependent inhibition of nuclear factor kappaB (NF-kappaB)-dependent genes, such as bcl-2, survivin, and cyclin D1, was also due, in part, to loss of Sp proteins. Curcumin also decreased bladder tumor growth in athymic nude mice bearing KU7 cells as xenografts and this was accompanied by decreased Sp1, Sp3, and Sp4 protein levels in tumors. These results show for the first time that one of the underlying mechanisms of action of curcumin as a cancer chemotherapeutic agent is due, in part, to decreased expression of Sp transcription factors in bladder cancer cells.

A NOVEL ACTIVATOR OF THE ORPHAN NUCLEAR RECEPTOR Nurr1 INHIBITS BLADDER TUMOR GROWTH

1524 Introduction and Objective: Nurr1 is an orphan nuclear receptor and a member of the nerve growth factor-I-B subfamily of the nuclear receptor family of transcription factors with no known endogenous ligand, or stimulator. We identified, for the first time, evidence that Nurr1 is expressed in a panel of human bladder cancer cells including 253J-BV, 253J-P, KU7, UM-UC3, UM-UC5, UM-UC6, UM-UC9, UM-UC10, UM-UC-13, and UM-UC14 cells. We then identified a new class of methylene-substituted diindolylmethanes (C-DIMs) which activate the ligand binding domain of Nurr1. Herein, we show the findings that Nurr1activation induces apoptosis in bladder cancer. Methods: To determine compound specificity, KU7 cells transfected with GAL4-Nurr1/pGAL4 were treated with varying concentrations of a panel of compounds, and luciferase activity was determined. 253J-BV, 253J-P, and KU7 cells were investigated for anti-proliferative response by MTT assay. Factors involved in apoptosis were evaluated by western blot. For in vivo bioluminescence study, 253J-BV cells labeled with a firefly gene were inoculated into mice bladder. Mice were treated with Nurr1 activator intra-peritoneally, and imaged weekly for their tumor size. Results: Activation of Nurr1 resulted in decreased cell survival and induction of cell death pathways, including DNA fragmentation, and poly (ADP-ribose) polymerase (PARP) cleavage. The specificity of the Nurr1-active compound was demonstrated using RNA interference in 253J-BV cells, whereby siRNA against Nurr1 attenuated ligand-dependent transactivation and PARP cleavage. Furthermore, activation of Nurr1 resulted in stimulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and siRNA experiments attenuated TRAIL production. In an orthotopic model of bladder cancer in nude mice, administration of a Nurr1-active C-DIM suppressed bladder cancer growth. Conclusions: These results identify Nurr1 as a potential target for bladder cancer therapy and also identify a Nurr1-active compound as a novel agent for activating this orphan receptor.

E-CADHERIN EXPRESSION POTENTIATES ERLOTINIB TREATMENT IN BLADDER CANCER

1080 E-CADHERIN EXPRESSION POTENTIATES ERLOTINIB TREATMENT IN BLADDER CANCER Teruo Inamoto*, Peter C Black, Mark B Fisher, Paul K Hegarty, Ameeta Arora, Liana Adam, David J McConkey, Colin P Dinney. Houston, TX. INTRODUCTION AND OBJECTIVE: Erlotinib (Tarceva®) is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor which inhibits cancer growth via the EGFR at nanomolar concentrations. Epithelial to mesenchymal transition (EMT) is emerging as a biomarker of response to EGFR tyrosine kinase inhibitors in lung cancer. We therefore tested whether E-cadherin expression levels altered erlotinib sensitivity in urothelial carcinoma. METHODS: The antiproliferative effect of erlotinib in the urothelial carcinoma cell lines 253J B-V, 253J P, UM-UC5, UM-UC10, UM-UC14, UM-UC9, UM-UC3, KU7 and UM-UC13 was measured by 3H-thymidine incorporation. E-cadherin expression was examined by western blot analysis and suppressed by a siRNA oligonucleotide. E-cadherin expression was enhanced by maintaining cells in an anchorage-independent culture in a poly-HEMA coated dish. RESULTS: 253J B-V, UM-UC5 and 253J P cells demonstracted low IC50 values (nM) for erlotinib of 3.1x10-8, 6.9x10-8 and 7.9x10-8 nM, respectively. UM-UC10, UM-UC14 and UM-UC9 cells demonstrated intermediate IC50 values of 1.6x10-7, 3.4x10-7, and 4.8 x10-6 nM, respectively. Fifty percent growth inhibition was not reached in UMUC3, KU7 and UM-UC13 cells so that these were considered erlotinib resistant. All of the cell lines that responded to erlotinib treatment expressed E-cadherin, whereas the three resistant cell lines did not. Depletion of endogenous E-cadherin in 253J B-V and UM-UC14 cells

Schedule Dependent Efficacy of Gefitinib and Docetaxel for Bladder Cancer

We determined the sequence specific efficacy of gefitinib and docetaxel treatment for bladder cancer. This combination was selected because it is currently under study in a phase II clinical trial. In vitro antiproliferative effects of gefitinib, docetaxel and a combination were determined in the 4 bladder cancer cell lines 253J B-V, UM-UC-3, KU-7 and UM-UC-13 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle analysis was analyzed using flow cytometry and propidium iodide labeling. Epidermal growth factor receptor downstream signaling was assessed by Western blot analysis. In vivo nude mice were injected subcutaneously with 253J B-V cells and treated with placebo, gefitinib, docetaxel, docetaxel followed by gefitinib or gefitinib followed by docetaxel. Tumor kinetics were established. Gefitinib demonstrated antiproliferative effect against 253J B-V cells (50% inhibitory concentration less than 0.5 muM) but no apoptotic effect in vitro, whereas docetaxel demonstrated antiproliferative and apoptotic effects. When gefitinib and docetaxel were combined, gefitinib enhanced the apoptotic and antiproliferative effects of docetaxel only when gefitinib was administered following docetaxel pretreatment. Apoptosis increased from 45% to 66%. In vivo there were significant differences in tumor weight in mice treated with combination therapy vs gefitinib or docetaxel alone. Importantly improved efficacy was observed when docetaxel was followed by gefitinib administration compared with gefitinib followed by docetaxel (mean tumor weight 42 vs 93 mg, p = 0.022). Sequence specific efficacy was not observed in UM-UC-3, UM-UC-13 and KU-7 cells, which are resistant to gefitinib. Docetaxel followed by gefitinib demonstrated sequence specific efficacy against gefitinib sensitive bladder cancer compared with gefitinib followed by docetaxel or either drug alone. Accordingly gefitinib administration concurrently or after chemotherapy might be the sequence of choice and it should be considered for future clinical trials.

Inhibition of Bladder Tumor Growth by 1,1-Bis(3′-Indolyl)-1-(p-Substitutedphenyl)Methanes: A New Class of Peroxisome Proliferator-Activated Receptor γ Agonists

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C-pPhC6H5) substituents have been identified as a new class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists that exhibit antitumorigenic activity. The PPARgamma-active C-DIMs have not previously been studied against bladder cancer. We investigated the effects of the PPARgamma-active C-DIMs on bladder cancer cells in vitro and bladder tumors in vivo. In this study, the PPARgamma-active compounds inhibited the proliferation of KU7 and 253J-BV bladder cancer cells, and the corresponding IC50 values were 5 to 10 and 1 to 5 micromol/L, respectively. In the less responsive KU7 cells, the PPARgamma agonists induced caveolin-1 and p21 expression but no changes in cyclin D1 or p27; in 253J-BV cells, the PPARgamma agonists did not affect caveolin-1, cyclin D1, or p27 expression but induced p21 protein. In KU7 cells, induction of caveolin-1 by each of the PPARgamma agonists was significantly down-regulated after cotreatment with the PPARgamma antagonist GW9662. DIM-C-pPhCF3 (60 mg/kg thrice a week for 4 weeks) inhibited the growth of implanted KU7 orthotopic and s.c. tumors by 32% and 60%, respectively, and produced a corresponding decrease in proliferation index. Treatment of KU7 cells with DIM-C-pPhCF3 also elevated caveolin-1 expression by 25% to 30%, suggesting a role for this protein in mediating the antitumorigenic activity of DIM-C-pPhCF3 in bladder cancer.